JP2019198320A - Hybrid yeast strain for bread production - Google Patents

Abstract

To provide practical yeasts for bread production that can produce breads with better flavors and shapes, and to provide a method for producing bread using the yeast.SOLUTION: By hybridizing Saccharomyces mikatae AK40 strain with Saccharomyces cerevisiae H24U1M strain, a practical bakery yeast that can produce bread with better flavor and shape can be obtained, and high-quality breads with more favorable flavor and shape can be produced by using the yeast in bread production.SELECTED DRAWING: None

Description

  The present invention relates to a hybrid yeast strain characterized in that a baked product having a good flavor can be obtained by using it for bread-making.

  In bread making, yeast plays an important role in expanding the bread dough with carbon dioxide generated when fermenting sugar and at the same time creating a unique flavor. Since such yeast products available for breadmaking have been selected based on the traits of bread dough fermentation ability, almost all strains are classified as the species Saccharomyces cerevisiae. However, since these are the same species, their traits are uniform and it is not easy to seek a differentiated flavor.

  As a method for improving the flavor of bread so far, modification of the production process (Patent Document 1), blending of auxiliary materials (Patent Document 2), addition of fermentation flavor liquid (Patent Document 3), yeast strain isolated from the natural world (Patent document 4), application of a yeast mutant imparted with drug resistance (patent document 5), etc., and it is said that there is some effect, but it is unclear whether that is enough.

  In such a technical background, there has been a demand in the art for the development of bakery yeast having unprecedented properties and characteristics that can produce breads with a better flavor.

  The genus Saccharomyces contains at least eight species, and strains classified exclusively into Saccharomyces cerevisiae are used in industries such as brewing. Of these, Saccharomyces mikatae (Saccharomyces mikatae) is a maltose non-fermentative wild species found in Japan and has not been used in the industry. However, a hybrid strain with Saccharomyces cerevisiae is used for winemaking. Then, it is reported that the product from which an aromatic component differs was obtained (nonpatent literature 1). However, a hybrid of Saccharomyces mikatae and Saccharomyces cerevisiae has never been applied to breadmaking so far.

JP2015-165777A Japanese Patent Laying-Open No. 2015-37393 Japanese Patent Laying-Open No. 2015-173633 JP 2012-191851 A Japanese Patent Application Laid-Open No. 2002-253211

Plos One, 8, e620533, 2013

  An object of the present invention is to newly develop a yeast strain from which a baked product exhibiting a good flavor can be obtained by being used for breadmaking. Furthermore, in terms of quality, especially flavor, aroma, and taste, while maintaining or exerting the fermentation power equivalent to or higher than that of conventional yeast, or in some cases, even at the expense of some fermentation power. It is an object of the present invention to newly produce a yeast strain capable of producing extremely excellent high-quality bread.

  In order to achieve the above-mentioned object, a strain having the characteristic excellent traits of Saccharomyces mikatae and Saccharomyces cerevisiae may be produced. As a result of intensive research on these points, the present inventors have been able to produce a yeast strain capable of obtaining a baked product having a characteristic and good flavor when used in breadmaking by crossing Saccharomyces mikatae and Saccharomyces cerevisiae. And the present invention was completed.

That is, the embodiment of the present invention is exemplified as follows.
(1) Baking yeast Saccharomyces sp. DHM15 strain (NITE P-02672).
(2) Baking yeast Saccharomyces sp. HM40 strain (NITE P-02938).
(3) A bread dough containing the yeast for baking according to (1) or (2).
(4) Saccharomyces mikatae AK40 strain (NITE P-02673) and Saccharomyces cerevisiae H24U1M strain (NITE P-02508) The production method of the yeast for bread making which can manufacture the bread | bread which has a more favorable flavor which belongs to (2).
(5) The method for producing a yeast for baking according to (4), wherein the hybridization is any of cell-to-spore junction, rare junction, or cell fusion.

  According to the present invention, by crossing the Saccharomyces mikatae AK40 strain and the Saccharomyces cerevisiae H24U1M strain, breads having a very good flavor can be produced and fermentation that can withstand the actual use of bread. A high-quality bread can be produced by producing a yeast strain for bread making having a high power and using the yeast strain for bread production.

It is a bar graph which shows the result obtained by using GC-MS about the bread | pan produced using the strain which concerns on this invention, and those parent strains, respectively, and analyzing an aroma component. In the figure, each bar graph sequentially represents isobutyl alcohol, isopentyl alcohol, acetoin, nonanal, ethyl caprylate, acetic acid, ethylhexanol, and phenylethyl alcohol from bottom to top.

  In the present invention, Saccharomyces mikatae AK40 strain which is a wild strain isolated from blueberry-like fruits and leaves in Tokachi region of Hokkaido, and a haploid strain H24 which shows a zygote type α derived from a commercial baker's yeast strain Saccharomyces cerevisiae. Then, the H24U1M strain, which is a uracil-requiring mutant strain obtained by the method of Kitamoto (Japan Brewing Association Magazine, 84 (12), 849-853, 1989), is crossed.

Using these as parent strains (AK40 strain, H24U1M), spore-to-cell junction and rare junction are performed to produce hybrid strains (DHM15, HM40), respectively. The spore-to-cell junction means a method of selecting a diploid hybrid strain formed by the heterozygous junction between a yeast spore (haploid) and a yeast haploid vegetative cell. Conjugation means a method of selecting a triploid hybrid strain produced by crossing a-zygous cells or α-zygous cells and haploid vegetative cells generated in diploid vegetative cells of yeast.

  The procedure for spore-to-cell junction is as follows. When AK40 is cultured in SPO agar medium (potassium acetate 1.0%, dry yeast extract 0.1%, glucose 0.05%, agar 2.0%) at 30 ° C. for 6 days, spores are formed. Then, it is a homothallic strain that self-diploidizes between cells that have been divided and transformed. On the other hand, H24U1M is a heterothallic strain in which mating type a does not convert and mates with cells of mating type α. Therefore, spore-formed AK40 cells were treated with a cell wall lytic enzyme and placed on one side of a YPD agar medium (dry yeast extract 1.0%, high polypeptone 2.0%, glucose 2.0%, agar 2.0%). The other side was coated with H24U1M. Under the microscope, 24 pairs of one AK40 spore and one H24U1M cell taken out by a micromanipulator were prepared and cultured at 30 ° C. for 2 days, then at 30 ° C. for 2 days. The 24 colonies that emerged were inoculated into minimal glucose agar medium (Yeast nitrogen base without amino acids 0.67%, glucose 2.0%, agar 2.0%) and cultured at 30 ° C. for 2 days. The cells were transplanted to an agar plate medium (Yeast nitrogen base without amino acids 0.67%, maltose 2.0%, agar 2.0%). When this was cultured in an anaerobic box at 30 ° C. for 1 day, 18 colonies grew, and one of them was purely isolated as a hybrid diploid DHM15 by spore-to-cell junction.

  On the other hand, the procedure of rare joining is as follows. AK40 may be a diploid of conjugated a / α, but a / a or α / α cells appear at a very low frequency in the process of cell division. Such cells can be conjugated to cells that exhibit the opposite conjugated type. Therefore, for both AK40 and H24U1M, the cells of one platinum loop were placed in a YPD medium (dry yeast extract 1.0%, high polypeptone 2.0%, in a test tube (diameter 1.8 cm × length 10.5 cm), 3 ml of glucose (2.0%) was inoculated and cultured at 30 ° C. with shaking (150 rpm). After 24 hours, 1 ml of the culture solution was aseptically centrifuged and the cells collected were washed twice with sterile water. This microbial cell was suspended in 6 ml of liquid minimal maltose medium (0.67% of yeast nitrogen base without amino acids, 2.0% of maltose), and statically cultured at 30 ° C. for 3 days. When 0.06 ml of this culture broth was inoculated into another new liquid minimal maltose medium and the culture was allowed to stand for 2 days. The grown yeast cells in this culture solution were streaked on a minimal maltose agar plate medium, and a hybrid HM40 (NITE P-02938) by rare mating was purely isolated.

The spore-to-cell mating strain DHM15 (Accession No. NITE P-02672) exhibits the following properties.
(1) Morphological properties When cultured at 30 ° C for 1 day in YPD medium (dry yeast extract 1.0%, high polypeptone 2.0%, glucose 2.0%), the cells are spherical or elliptical and large. The length is 4-7 μm × 5-9 μm, and sprouting multipolar. In addition, colonies when cultured for 1 day at 30 ° C. on a YPD agar medium are light brown and shiny. In addition, spore formation is observed when cultured on an SPO agar medium at 30 ° C. for 6 days.
(2) Physiological properties Grows at 20 to 37 ° C.
(3) Fermentable glucose of sugar: +
Galactose: +
Sucrose: +
Maltose: +
Lactose:-
Raffinose: +
Trehalose: +
Melibiose: +
(4) Carbon-assimilating glucose of carbon source: ++
Galactose: ++
L-sorbose:-
Sucrose: ++
Maltose: ++
Cellobiose:-
Trehalose: ++
Lactose:-
Melibiose: ++
Raffinose: +
Merezitose: ++
Inulin:-
Soluble starch:-
D-xylose:-
L-arabinose:-
D-arabinose:-
D-ribose:-
L-rhamnose:-
Ribitol:-
D-mannitol:-
Glycerol:-
Ethanol: ++
α-methylglucoside: ++
Salicin:-
Succinic acid:-
Citric acid:-
Myo-inositol:-
D-Glucosamine:-

Rare mating hybrid HM40 (Accession No. NITE P-02938) exhibits the following properties.
(1) Morphological properties When cultured at 30 ° C for 1 day in YPD medium (dry yeast extract 1.0%, high polypeptone 2.0%, glucose 2.0%), the cells are spherical or elliptical and large. The length is 4-7 μm × 5-9 μm, and sprouting multipolar. In addition, colonies when cultured for 1 day at 30 ° C. on a YPD agar medium are light brown and shiny. In addition, spore formation is observed when cultured on an SPO agar medium at 30 ° C. for 6 days.
(2) Physiological properties Grows at 20 to 37 ° C.
(3) Fermentable glucose of sugar: +
Galactose: +
Sucrose: +
Maltose: +
Lactose:-
Raffinose: +
Trehalose:-
Melibiose: +
(4) Carbon-assimilating glucose of carbon source: ++
Galactose: ++
L-sorbose:-
Sucrose: ++
Maltose: ++
Cellobiose:-
Trehalose: ++
Lactose:-
Melibiose: ++
Raffinose: +
Merezitose: ++
Inulin:-
Soluble starch:-
D-xylose:-
L-arabinose:-
D-arabinose:-
D-ribose:-
L-rhamnose:-
Ribitol:-
D-mannitol:-
Glycerol:-
Ethanol: ++
α-methylglucoside: ++
Salicin:-
Succinic acid:-
Citric acid:-
Myo-inositol:-
D-Glucosamine:-

The hybrid parent strain AK40 (accession number NITE P-02673) exhibits the following properties.
(1) Morphological properties When cultured at 30 ° C. for 1 day in a YPD medium, the cells are spherical or elliptical and have a size of 6-8 μm × 5-6 μm and sprouting multipolarly. In addition, colonies when cultured for 1 day at 30 ° C. on a YPD agar medium are light brown and shiny. In addition, spore formation is observed when cultured on an SPO agar medium at 30 ° C. for 6 days.
(2) Physiological properties Grows at 20 to 37 ° C.
(3) Fermentable glucose of sugar: +
Galactose: +
Sucrose: +
Maltose:-
Lactose:-
Raffinose: +
Trehalose:-
Melibiose: +
(4) Carbon-assimilating glucose of carbon source: ++
Galactose: ++
L-sorbose:-
Sucrose: ++
Maltose: ++
Cellobiose:-
Trehalose: +
Lactose:-
Melibiose:-
Raffinose: +
Merezitose: ++
Inulin:-
Soluble starch:-
D-xylose:-
L-arabinose:-
D-arabinose:-
D-ribose:-
L-rhamnose:-
Ribitol:-
D-mannitol: ++
Glycerol:-
Ethanol: ++
α-methylglucoside: ++
Salicin:-
Succinic acid:-
Citric acid:-
Myo-inositol:-
D-Glucosamine:-

The other parent strain H24U1M (accession number NITE P-02508) of the cross shows the following properties.
(1) Morphological properties When cultured at 30 ° C. for 1 day in a YPD medium, the cells are spherical or elliptical, have a size of 4-6 μm × 3-5 μm, and sprouting multipolarly. In addition, colonies when cultured for 1 day at 30 ° C. on a YPD agar medium are light brown and shiny. In addition, no spore formation was observed even when cultured on an SPO agar medium at 25 ° C. for 7 days.
(2) Physiological properties Grows at 20 to 37 ° C.
(3) Fermentable glucose of sugar: +
Galactose: +
Sucrose: +
Maltose: +
Lactose:-
Raffinose: +
Trehalose:-
Melibiose:-
(4) Carbon-assimilating glucose of carbon source: ++
Galactose: ++
L-sorbose:-
Sucrose: ++
Maltose: ++
Cellobiose:-
Trehalose: +
Lactose:-
Melibiose:-
Raffinose: +
Merezitose: +
Inulin:-
Soluble starch:-
D-xylose:-
Citric acid:-
Myo-inositol:-
D-Glucosamine:-

These AK40 strain, H24U1M strain, DHM15 strain, and HM40 strain are all incorporated in the National Institute of Technology and Evaluation Microorganism Depositary Center (Kazusa Kamashi 2-5-8, Kisarazu City, Chiba Prefecture, Japan 292-0818). The AK40 strain was deposited on April 4, 2018, the H24U1M strain was deposited on July 14, 2017, and the DHM15 strain was deposited on April 4, 2018. The deposit numbers are NITE P-02673, respectively. NITE P-02508 and NITE P-02672. The HM40 strain has also been deposited with the Patent Microorganism Deposit Center on April 15, 2019, and its deposit number is NITE P-02938.

Examples of the present invention will be described below, but the present invention is not limited to these examples, and various modifications can be made within the technical idea of the present invention.

  The bread dough fermenting power of the hybrid strains DHM15 and HM40 of the present invention was compared to Saccharomyces mikatae AK40 strain (crossbred parent), Saccharomyces cerevisiae H24U1M (the other crossbred parent) and Saccharomyces cerevisiae H strain 46 comparative strain H.

Each strain was cultured in 10 ml of YPD medium in a 50 ml Erlenmeyer flask by reciprocal shaking culture (150 rpm) at 30 ° C. for 24 hours. Peptone 4.0%, KH ₂ PO ₄ 0.2%, MgSO ₄ .7H ₂ O 0.1%, NaCl 2.0%, adecanol LG-2940.05%, sucrose 2.0%) Rotating shaking culture (150 rpm) at 30 ° C. for a time. The cultured cells were collected by centrifugation, washed twice with distilled water, and then placed on a dried absorbent plate for several minutes to obtain cultured cells. Although the solid content of the cultured microbial cells is about 30%, an accurate numerical value was calculated by drying a part of the cultured microbial cells. In the following experiments, the cultured microbial cells were used for preparing the dough as a weight converted to a solid content of 33%.

1 suspension of 1.0 g of a suspension containing 10 g of wheat flour (strong), 5.5 ml of distilled water or 5.5 ml of distilled water containing 0.5 g of sucrose and 0.2 g of NaCl and 0.2 g of yeast cells (solid content 33%) Kneaded for minutes to prepare medium dough or low sugar dough, respectively. These doughs are put in a 2.4 cm × 20 cm test tube, the amount of carbon dioxide generated is guided to a graduated cylinder in saturated saline, and the amount of carbon dioxide generated per hour at 30 ° C. is used as the bread dough fermentation power. It was measured.

As shown in Table 1, compared to HP467, DHM15 outperforms medium seed dough fermentation power and is equivalent in low sugar dough fermentation power, so that AK40's bread dough fermentation power crosses with H24U1M. As a result, it was found that it was greatly improved. On the other hand, the bread dough fermenting power of the hybrid strain HM40 was not as high as that of HP467, but was higher than that of AK40, and it was found that it was improved by crossing with H24U1M.

Using the hybrid strain DHM15 prepared in the same manner as in Example 1 and the commercially available baker's yeast isolate HP467, bread was produced by the medium seed method, and their quality was compared.

210g of wheat flour (strong), 6.0g of yeast cultured cells (33% solids), 0.15ml (20mg / ml) ascorbic acid solution and 126ml distilled water for 3 minutes with a pin mixer A medium seed dough was prepared so as to be 24.0 ± 1.0 ° C. When this was fermented at 30 ° C. for 4.5 hours, 90 g of flour, 15.0 g of sugar, 6.0 g of salt, 15.0 g of shortening, and 75 ml of distilled water were added, and the mixture was kneaded for about 4 minutes, and kneaded. The main body dough was prepared so that the temperature was 30.0 ± 0.5 ° C. Further, after a floor time of 30 minutes at 30 ° C., the dough was divided by hand by 100 g and rounded to take a bench time of 15 minutes at 30 ° C. This was molded with a molder and subjected to final fermentation at 38 ° C. and 85% humidity for 55 minutes, followed by baking at 180 ° C. for 25 minutes. This was allowed to cool at room temperature, and the specific volume was calculated by measuring the weight and volume. The results are shown in Table 2 (Breadmaking test results). Although the specific volume of bread made with DHM15 was lower than HP467, it was a value that could be sufficiently evaluated at 5.0 or higher.

Next, the produced bread was evaluated for volume, shape, baked color, internal shape, softness, hue, fragrance and taste by a panel of 11 persons. Table 3 (sensory evaluation results) shows the results of setting the result of bread made with HP467, which is a control plot, to 50. Bread made with DHM15 had an evaluation equal to or higher than that for items other than volume, and was particularly excellent in terms of aroma and taste.

Using the hybrid HM40 and the commercial baker's yeast isolate HP467 in the same manner as in Example 2, bread was prepared by the medium seed method, and their quality was compared.

The results are shown in Table 4 (Breadmaking test results). As is clear from the table, the specific volume of bread made with HM40 was lower than HP467, but was a value that could be sufficiently evaluated at 5.0 or higher.

In the same manner as in Example 2, the produced bread was evaluated for volume, shape, baked color, internal shape, softness, hue, fragrance and taste using a panel of 10 people. Table 5 (sensory test results) shows the results of setting the result of bread made with HP467, which is a control group, to 50. Bread made with HM40 had the same or higher evaluation in terms of items other than volume, and was particularly superior in terms of fragrance and taste.

  The dough-fermenting power of Saccharomyces cross-species hybrid strains and the aroma components of bread produced using them were analyzed.

<Used strain>
As the strain used, the strain described in Table 6 below was used.

<Fabric fermentation power>
The dough fermenting power was measured with a dough containing 0% sugar according to the yeast industry association method. The results are shown in Table 7 below (the unit of the numerical value is ml), and it was confirmed that the DHM15 strain and the HM40 strain have the same or higher 0% dough fermentation power than the control.

<Aroma component>
Using these strains, bread was produced by a 70% medium seed method. The crumb portion of the obtained bread was subjected to GC-MS under the following conditions to analyze the aroma components.

○ Apparatus Shimadzu GC-MS-QP2010Ultra
○ Aroma component collection condition Sample: 4g of bread crumb
Collection material: SPME fiber (100um PDMS, Fused Silica 24Ga)
Collection time: 30 minutes, collection temperature 70 ° C
○ GC-MS analysis conditions Column: TC-WAX (0.25 mm, ID × 60 m), 40 ° C./2 min → 240 ° C. (10 min)
Ionization method: EI
Internal standard: cyclohexanol

  The obtained results are shown in Table 8 and FIG. From these results, the ratio of the main aroma components is significantly different from that of the control, and it is clear from the results of the sensory test of the bread that has already been described (most panelists felt a clear scent difference from the control). Moreover, the scent of the bread produced using the hybrid baker's yeast strain according to the present invention was very good.

  In the figure, the vertical axis represents the peak area and the horizontal axis represents the strain. The strains represent, in order from the left, our general yeast (HP467), AK40, H24U1M, DHM15, and HM40. Each bar graph indicates, in order from the bottom, isobutyl alcohol, isopentyl alcohol, acetoin, nonanal, ethyl caprylate, acetic acid, ethylhexanol, and phenylethyl alcohol.

The present invention is summarized as follows.

An object of the present invention is to provide a practical bread-making yeast capable of producing breads with better flavor and shape, a method for producing breads using the yeasts, and the like.

Then, by crossing Saccharomyces mikatae AK40 strain with Saccharomyces cerevisiae H24U1M strain, a practical bread-making yeast that can produce breads with better flavor and shape. It can be obtained, and by using the yeast for bread production, it is possible to produce high-quality breads with a more favorable flavor and shape, particularly breads with excellent flavor, aroma and taste.

The deposit number of the microorganism deposited in the present invention is shown below.
(1) Saccharomyces mikatae AK40 strain (NITE P-02673)
(2) Saccharomyces cerevisiae H24U1M strain (NITE P-02508)
(3) Saccharomyces sp. DHM15 strain (NITE P-02672)
(4) Saccharomyces sp. HM40 strain (NITE P-02938)

Claims (5)

Baking yeast Saccharomyces sp. DHM15 strain (NITE P-02672). Baking yeast Saccharomyces sp. HM40 strain (NITE P-02938). Bread dough containing the yeast for breadmaking according to claim 1 or 2. Saccharomyces micatae AK40 strain (NITE P-02673) and Saccharomyces cerevisiae H24U1M strain (NITE P-02508) are crossed with the genus S. A method for producing a yeast for baking, which can produce bread having a better flavor. The method for producing a yeast for baking according to claim 4, wherein the crossing is one of cell-to-spore junction, rare junction, or cell fusion.

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